Insulin and diabetes

how can blood glucose levels be measured

enzymatically from a drop of blood with glucose oxidase, H₂O₂ is detected colorimetrically

glucose + O₂ → glucose-δ-lactone + H₂O₂

longer term average inferred measuring haemoglobin glycation

reaction of glucose with primary amines in Hb, rate of glycation is proportional to [glucose]

how does increase in blood glucose lead to secretion of insulin

insulin is secreted when intra-cellular ATP levels rise in pancreatic B-cells, sign of overflow of its energy status

more glucose, more respiration, more ATP produced

ATP:ADP ratio increases and promotes influx of Ca²⁺ into cell which induces synthesis and release of insulin

how is insulin made

as a precursor

pro-insulin is cleaved in 2 places to give A and B chain

insulin has 2 intermolecular disulphide bonds between the chains and 1 intramolecular disulphide bond on the A chain

how is insulin stored

intracellular granules as crystals formed from hexamers

hexamer surrounds 2 Zn²⁺ ions and loses these ions when it breaks down into a dimer, then monomer

insulin receptor (IR)

a2-B2 tetramer with long extracellular domain to bind insulin and intracellular TK domain

insulin binds a-subunits and structure of IR changes to bring TK domains together

B-subunits autophosphorylate 3 tyrosines which transduces signal into cell by turning on receptor’s catalytic activtiy

activated receptor phosphorylates multiple intracellular proteins which alters their activity and leads to biological response

autophosphorylation of 3 tyrosines

they are in a loop, and when phosphorylated, the loop moves and the active site is open to bind substrates

how does insulin increase glucose uptake into cells

in absence of insulin, GLUT4 glucose transporters are in cytoplasmic vesicles

binding of insulin to IR leads to phosphorylation of IRS, causing several phosphorylation cascades and leading to rapid fusion of vesicles with cell membrane, GLUT4 is in the membrane to facilitate glucose uptake

when insulin levels decrease and IRs are unoccupied, GLUT4 are recycled back into vesicles

how does insulin signalling lead to glycogen synthesis

insulin binds IR, IRS is phosphorylated by IR, causes several phosphorylation cascades including

phosphorylation (activation) of glycogen synthase phosphatase

this dephosphorylates glycogen synthase (activating it) which synthesises glycogen

type 1 diabetes mellitus

autoimmune, usually arising in childhood

body destroys insulin/cells that make it

destruction of pancreatic islet B-cells by T cells leads to insulin deficiency

type 2 diabetes mellitus

characterised by high blood and urine glucose levels that are resistant to insulin

tissues do not respond to circulating insulin

unable to push plasma glucose into storage or metabolise into fat even with high insulin

pancreas keeps pumping out insulin, leading to other issues

fat disposed in adipose tissue, muscle, liver, pancreas, heart etc

pathological outcomes of lack of insulin signalling

high blood/urine glucose levels, lack of glucose uptake causes cellular starvation

cells breakdown fats to generate energy and produce ketone byproducts

ketones build up and make blood acidic

ketoacidosis is accompanied by water and ion loss and thick blood - diabetic coma and life-threatening

ketone bodies

acetoacetate and hydroxybutyrate

soluble forms of energy to be released into bloodstream

how blood glucose and insulin concentrations change during and between meals

glucose levels spike at meals, closely followed by insulin, neither levels return to 0

injected insulin acts like insulin but levels reach 0 between meals

pathogenic glucose levels are consistently high

fast acting insulins

immediate, shift insulin towards active monomer by disruption of dimer interface

insulin Aspart

insulin LisPro

insulin Aspart

mutation in B28 from Pro to Asp

disrupts dimer by opposed negative charges with 21Glu

no dimer, no hexamer, so more of the active form

insulin LisPro

mutation in B28-29 from Pro28-Lys29 to Lys28-Pro29

long acting insulins

avoid cellular hypoglycaemia (glucose starvation) overnight

add fatty acid to end of B chain at Lys29 (myristoylated Lys29)

increases pI to reduce solubility/diffusion

adding residues with high pKas slow diffusion

semi-synthesis of insulin

from pig insulin

cleave Ala30 from pig insulin by specific proteolysis

add specific peptide using trypsin in organic solvent, protect Lys NH2 and add Thr or another residue using solid state synthesis

deprotection